Journal articles

Analogues of the Ca2+-releasing intracellular messenger d-myo-inositol 1,4,5-trisphosphate [1, Ins(1,4,5)P3] are important synthetic targets. Replacement of the α-glucopyranosyl motif in the natural product mimic adenophostin 2 by d-chiro-inositol in d-chiro-inositol adenophostin 4 increased the potency. Similar modification of the non-nucleotide Ins(1,4,5)P3 mimic ribophostin 6 may increase the activity.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are widely expressed intracellular channels that release Ca2+ from the endoplasmic reticulum (ER). We review how studies of IP3Rs removed from their intracellular environment (‘ex cellula’), alongside similar analyses of ryanodine receptors, have contributed to understanding IP3R behaviour. Analyses of permeabilized cells demonstrated that the ER is the major intracellular Ca2+ store, and that IP3 stimulates Ca2+ release from it.

Fluorescence polarization (FP) can be used to measure binding of a small fluorescent ligand to a larger protein because the ligand rotates more rapidly in its free form than when bound. When excited with plane polarized light, the free fluorescent ligand emits depolarized light, which can be quantified. Upon binding, its rotation is reduced and more of the emitted light remains polarized. This allows FP to be used as a non-destructive assay of ligand binding.

In the 30 years since IP3 (inositol 1,4,5-trisphosphate) was first shown to release Ca2+ from intracellular stores, the importance of spatially organized interactions within IP3-regulated signalling pathways has been universally recognized. Recent evidence that addresses three different levels of the structural determinants of IP3-evoked Ca2+ signalling is described in the present review.

Fluorescence polarization (FP) allows quantification of the binding of a small fluorescent ligand to a larger protein because the free ligand rotates more rapidly than the bound form. This protocol describes an FP assay for the binding of fluorescein-labeled inositol 1,4,5-trisphosphate (IP3) to amino-terminal fragments of the IP3 receptor at different temperatures and in the presence of competing ligands. The method requires fluorescein-labeled IP3 and a plate-reader capable of FP measurements.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca(2+) channels. They are expressed in most animal cells and mediate release of Ca(2+) from the endoplasmic reticulum (ER) in response to the many stimuli that evoke formation of inositol 1,4,5-trisphosphate (IP3). The opening of individual IP3Rs causes small, transient, local increases in cytosolic Ca(2+) concentration, and these events are the fundamental units of Ca(2+) signaling.

Calcium-binding protein 1 (CaBP1) is a neuron-specific member of the calmodulin superfamily that regulates several Ca2+ channels, including inositol 1,4,5-trisphosphate receptors (InsP3Rs). CaBP1 alone does not affect InsP3R activity, but it inhibits InsP 3-evoked Ca2+ release by slowing the rate of InsP 3R opening. The inhibition is enhanced by Ca2+ binding to both the InsP3R and CaBP1. CaBP1 binds via its C lobe to the cytosolic N-terminal region (NT; residues 1-604) of InsP3R1.

IP(3)R (IP(3) [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca(2+) channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IP(3)R, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP(3)-evoked Ca(2+) release via IP(3)R1 and IP(3)R2, but inhibited IP(3)R3.

Binding of IP3 (inositol 1,4,5-trisphosphate) to the IP3-binding core (residues 224-604) of IP3Rs (IP3 receptors) initiates opening of these ubiquitous intracellular Ca2+ channels. The mechanisms are unresolved, but require conformational changes to pass through the suppressor domain (residues 1-223). A calmodulin-binding peptide derived from myosin light chain kinase uncouples these events.

Inositol-1,4,5-trisphosphate receptors (InsP 3Rs) and ryanodine receptors (RyRs) are tetrameric intracellular Ca 2+ channels. In each of these receptor families, the pore, which is formed by carboxy-terminal transmembrane domains, is regulated by signals that are detected by large cytosolic structures. InsP 3R gating is initiated by InsP 3binding to the InsP 3-binding core (IBC, residues 224-604 of InsP 3R1) and it requires the suppressor domain (SD, residues 1-223 of InsP 3R1).